The long-term objectives of this project are: 1) to understand the molecular mechanisms responsible for DNA sequence amplification; 2) to understand the relationships between nuclear architecture and function in the mammalian genome; and 3) to determine whether amplification disturbs these relationships. We have isolated overlapping recombinant cosmids representing more than 500 kb of contiguous DNA sequence from the amplified dihydrofolate reductase (DHFR) domain of a methotrexate-resistant Chinese hamster cell line. We have identified and characterized three origins of replication, three termini (at least two of which may be fixed) , four active genes, and several matrix attachment sites within this large domain. We have also used these clones to define rearrangements of the DHFR domain that accompany DHFR gene amplification in several independently isolated, moderately drug-resistant Chinese hamster cell lines. The specific aims of the proposed project are to: 1) isolate a series of hybridization probes positioned at about 20 MB intervals on the long arm of chromosome 2 (site of the DHFR locus); these probes will be used in pulse-field gradient gel electrophoretic and fluorescence in situ hybridization analyses in the studies outlined in aims 2&3; 2) analyze the earliest recoverable amplification events in both selected and spontaneous DHFR amplificants, and follow clonal derivatives of these amplificants to assess subsequent rearrangements; 3) isolate the progeny of the two daughter cells of initial amplification events, in order to determine whether these two cell types show the additive or reciprocal changes in DHFR gene copy number that are predicted by the overreplication or unequal sister chromatid exchange models, respectively; 4) precisely map the termini that we have previously localized in the 500 kb DHFR domain using a two-dimensional gel method; 5) determine whether the termini represent or map close to matrix-attachment sites; and 6) determine whether these termini are capable of arresting replication in one or both directions when introduced into an autonomously replicating plasmid.